Novel enzymatic methods to generate high yields of sequence specific rnas with extreme precision

ABSTRACT

Described herein are synthetic methods for producing sequence-specific RNA oligonucleotides that eliminate impurities produced in prior art methods. In one aspect, a first amplification primer includes one or more deoxyuridine residues, wherein at least one of the one or more deoxyuridine residues is at position −1, −2, −3, −4 or −5 of the promoter region for the single-subunit, DNA-dependent RNA polymerase. The deoxyuridines are excised to provide an amplified functional template DNA which is then used to synthesize RNA which has reduced immunogenic double stranded RNA compared to controls.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No. 16/587,563, filed on Apr. 24, 2020, which claims priority to U.S. Provisional Application 62/837,906 filed on Apr. 24, 2019, and U.S. Provisional Application 62/933,119, filed on Nov. 8, 2019, which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under MCB-1516896 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure is related to novel enzymatic methods of synthesizing product RNAs which reduce double stranded impurities produced in prior methods.

SEQUENCE LIST

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated by reference in its entirety. Said ASCII copy, created on Oct. 28, 2021 is named “UMA0129US3P Sequence List TXT” and is 3,002 bytes in size.

BACKGROUND

The 21^(st) century is seeing huge advances in RNA biology and biochemistry. These studies require pure, monodisperse preparations of RNA. In RNA applications, mRNA therapeutics are poised to become a major player in biologic pharmaceuticals, an already large, established, and growing field (indeed, it is argued that RNA biologics will largely replace protein biologics). The main challenge holding back applications is that the mRNA often triggers the innate immune response in patients, a potentially lethal outcome. It is well known that human cells have an innate immune response that responds to the presence of double stranded RNA (which the mRNA should not be, in theory).

RNA therapeutics approaches are hampered by an undesired immune response that has led to dramatic failures in clinical trials (including some patient deaths). Various companies have developed technologies built on the incorporation of base analogs into the RNA. Success with this approach has been promising, but limited. Indeed, mRNA drugs in clinical trials are vaccines, exploiting the immune response as an adjuvant. Their original target, gene replacement therapies, continue to languish, as extensive purification only sometimes alleviates this problem, and not fully.

Currently, there are two established routes to generate synthetic RNA: chemically or enzymatically. The chemical route is generally limited to RNAs shorter than about 30-50 bases in length, depending on the application. The enzymatic route is routinely used to generate large quantities of short (e.g., 30 base) and long (kilobase) RNA in vitro using T7 RNA polymerase (T7RP) or one of its closely related family members. T7RP is a promoter specific, highly processive enzyme. However, it also participates in promoter-independent transcription that results in unwanted product. This promiscuity often contaminates the transcription pool with undesired longer (and shorter) products. It is believed to be longer, double stranded impurities in the RNA that present the biggest challenge to the RNA therapeutics industry, as delivered RNAs often invoke an unwanted innate immune response (which has led to patient deaths in clinical trials).

What is needed are novel synthetic methods for sequence-specific RNA oligonucleotides that eliminate impurities produced in prior art methods.

BRIEF SUMMARY

A method of synthesizing RNA comprises

-   -   providing a starting functional template DNA for a         single-subunit, DNA-dependent RNA polymerase, and first and         second amplification primers for the starting functional         template DNA, wherein the starting functional template DNA         comprises a promoter region for the single-subunit,         DNA-dependent RNA polymerase,         -   wherein the first amplification primer is complementary to a             template strand of the template DNA and comprises one or             more deoxyuridine residues, wherein at least one of the one             or more deoxyuridine residues is at position −1, −2, −3, −4             or −5 of the promoter region for the single-subunit,             DNA-dependent RNA polymerase, and         -   wherein the second amplification primer is complementary to             a nontemplate strand of the template DNA;     -   amplifying the starting functional template DNA in the presence         of the first and second amplification primers and under         conditions for DNA amplification to provide an amplified         functional template DNA, wherein the amplified functional         template DNA comprises the one or more deoxyuridine residues at         position −1, −2, −3, −4 and/or −5 of the promoter region for the         single-subunit, DNA-dependent RNA polymerase;     -   excising the uracil bases of the deoxyuridine residues from the         promoter region of the amplified functional template DNA to         provide a modified, amplified functional template DNA;     -   carrying out a transcription reaction to synthesize RNA from the         modified, amplified functional template DNA in the presence of         the single-subunit, DNA-dependent RNA polymerase and under         conditions for RNA synthesis; and     -   isolating the synthesized RNA from the transcription reaction.

In another aspect, a method of synthesizing RNA, comprises

-   -   providing a starting functional template DNA for an RNA         polymerase and an amplification primer that is complementary to         a template strand of the template DNA and comprises one or more         deoxyuridine residues, wherein the starting functional template         DNA comprises a promoter region for RNA polymerase, and wherein         at least one of the one or more deoxyuridine residues is at         position −4 or −5 of a promoter region for the RNA polymerase;     -   amplifying the starting functional template DNA in the presence         of the amplification primer under conditions for DNA         amplification to provide an amplified functional template DNA,         wherein the amplified functional template DNA comprises the one         or more deoxyuridine residues at position −4 or −5 of the         promoter region for the RNA polymerase;     -   excising the uracil bases of the deoxyuridine residues from the         promoter region of the amplified functional template DNA to         provide a modified, amplified functional template DNA;     -   carrying out a transcription reaction to synthesize RNA from the         modified, amplified functional template DNA in the presence of         the RNA polymerase and under conditions for RNA synthesis to         provide a transcription reaction, wherein the polymerase is a T7         RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, or an         SP6 RNA polymerase; and     -   isolating the synthesized RNA from the transcription reaction.

In an aspect, a method of synthesizing a product RNA comprises

-   -   preparing a reaction mixture comprising a functional template         DNA for the product RNA, an RNA polymerase and a capture DNA         under conditions for product RNA synthesis,     -   synthesizing the product RNA in the reaction mixture,     -   optionally removing the capture DNA after product RNA synthesis,         and     -   optionally purifying the product RNA,     -   wherein the capture DNA is 3′ end protected, and is         complementary to 8 to 20, preferably 12 to 20 nucleotides at the         3′ end of the product RNA, and     -   wherein the functional template DNA, the RNA polymerase, capture         DNA or a combination thereof, is optionally covalently or         noncovalently attached to one or more solid supports.

In another aspect, a method of synthesizing a product RNA comprises

-   -   preparing a reaction mixture comprising a functional template         DNA for the product RNA and an RNA polymerase under conditions         for RNA synthesis, wherein the RNA polymerase is covalently or         noncovalently linked to the functional template DNA in a manner         that allows for synthesis of the product RNA,     -   synthesizing the product RNA in the reaction mixture, and     -   optionally purifying the product RNA,     -   wherein the functional template DNA, the RNA polymerase, or         both, is optionally covalently or noncovalently attached to a         solid support.

In another aspect, a method of synthesizing a product RNA comprises

-   -   preparing a reaction mixture comprising a functional template         DNA for the product RNA, and an RNA polymerase under conditions         for RNA synthesis, wherein the RNA polymerase is covalently or         noncovalently linked to a nontemplate DNA that is 10 to 10,000         nucleotides or longer in length, and wherein the nontemplate DNA         is complementary to a minimum of 10 nucleotides of the template         DNA upstream of the transcription start site, or wherein the         nontemplate DNA is complementary to all or part of the entire         template strand DNA in its coding region,     -   synthesizing the product RNA in the reaction mixture, and     -   optionally purifying the product RNA,     -   wherein the template DNA, the nontemplate, the RNA polymerase,         or a combination thereof, is optionally covalently or         noncovalently attached to a solid support.

In yet another aspect, a method of synthesizing a product RNA comprises

-   -   (i) preparing a reaction mixture comprising a functional         template DNA for the product RNA, and an RNA polymerase under         conditions for RNA synthesis, wherein functional template DNA         and the RNA polymerase are immobilized to a solid support, and         wherein the contacting is done in a flow chamber,     -   synthesizing the product RNA in the reaction mixture and         continuously removing the product RNA from the flow chamber         while flowing fresh RNA synthesis reagents into the flow         chamber, and     -   optionally purifying the product RNA,     -   or     -   (ii) preparing a reaction mixture comprising a functional         template DNA for the product RNA, and an RNA polymerase under         conditions for RNA synthesis, wherein the RNA polymerase is         covalently or noncovalently linked to a nontemplate DNA that is         10 to 10,000 nucleotides or longer in length, and wherein the         nontemplate DNA is complementary to a minimum of 10 nucleotides         of the template DNA upstream of the transcription start site, or         wherein the nontemplate DNA is complementary to all or part of         the entire template strand DNA in the coding region, wherein the         nontemplate DNA and the RNA polymerase are immobilized to a         solid support, and wherein the contacting is done in a flow         chamber,     -   synthesizing the product RNA and continuously removing the         product RNA from the flow chamber while flowing fresh RNA         synthesis reagents into the flow chamber, and     -   optionally purifying the product RNA.

In another aspect, a method of providing a modified, amplified functional template DNA comprises

-   -   providing a starting functional template DNA and an         amplification primer, the amplification primer comprising one or         more deoxyuridines on the nontemplate strand, under conditions         for DNA amplification,     -   amplifying the starting DNA functional template to provide an         amplified DNA functional template, and     -   excising the deoxyuridines from the amplification primer to         provide the modified, amplified functional template DNA.

In a yet further aspect, a method of purifying a product RNA, comprises

-   -   adding a capture DNA to a reaction mixture for producing the         product RNA, the reaction mixture comprising the product RNA, a         functional template DNA for the product RNA, and an RNA         polymerase under conditions for RNA synthesis; wherein the         capture DNA is 10 nucleotides or longer in length, and wherein         the capture DNA comprises a capture sequence complementary to a         minimum of 10 nucleotides of the 3′ terminus of the product RNA,         and wherein the capture DNA is tethered to a solid support;     -   incubating the tethered capture DNA and the reaction mixture to         provide binding of the capture DNA and the product RNA,     -   washing to remove unbound reaction components, and     -   eluting the product RNA from the tethered capture DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows high yield transcription favors impurities. On-pathway synthesis (and accumulation of large concentrations) of desired RNA (R_(n)) drives off-pathway primer extension that generates heterogeneous, double stranded impurities.

FIG. 2 shows inhibition of self-primed extension of synthetic RNA. FIG. 2A shows self-primed extension reaction of synthetic RNA. FIG. 2B shows denaturing (20% urea) gel electrophoretic analysis: radiolabeled 24-base synthetic RNA was reacted with T7 RNA polymerase for 0 min, 5 min and 4 h, in the absence (−) or presence (+) of capture DNA. FIG. 2C shows that DNA captures the 3′ end of the RNA, competing with self-primed extension.

FIG. 3 shows the length optimization of capture DNA. Denaturing gel analysis, in the absence and presence of 3′-modified complementary capture DNA oligonucleotides of lengths 9, 11, 14 and 17-bases. Reactions were carried out at 37° C.; approximate predicted melting temperatures are shown for the RNA/DNA duplexes. The sequence of the RNA is presented for reference.

FIG. 4 shows capture DNA eliminates self-primed extension during RNA synthesis. Presence of 3′-complementary capture DNA sequesters product RNA, inhibiting self-primed extension during transcription. FIG. 4A shows encoded RNAs and corresponding capture DNAs. FIG. 4B shows denaturing (20% urea) gel analysis of 4 h high yield transcription reactions. Transcripts were labeled by [α-³²P] ATP. Lanes “24” above demonstrate clearly that Capture-17 inhibits formation of primer extended products. Lanes “24Alt” show that for an RNA sequence that does not promote self-primed extension, the presence of Capture-17 has no effect. Finally, lanes “34” show that the effect is not dependent on the length of the RNA and that self-primed extension proceeds farther on longer RNAs.

FIG. 5 shows the effect of concentration/stoichiometry of capture DNA. Denaturing (20% urea) gel electrophoreses analysis. All transcripts were labeled by [α-³²P] ATP. FIG. 5A shows high yield transcription reaction (4 h) for RNA-24 in the presence of varying concentrations of complementary capture DNA (Capture-17). FIG. 5B shows time dependence of transcription reaction products in the presence (+) or absence (−) of 200 μM capture DNA.

FIG. 6 shows increasing product purity with increasing salt in the reaction.

FIG. 7 illustrates an embodiment of tethered, high salt transcription. FIG. 7A shows site-specific coupling of nontemplate DNA to protein generates a “universal reagent,” to which template DNA of arbitrary RNA sequence can be added to generate a functional template encoding that RNA sequence. FIG. 7B shows to prevent primer extension reactions, salt was added, as indicated, to each reaction prior to initiation of high yield transcription (3 hrs, 7.5 mM each substrate NTP). The crosslinked species generates substantially more pure, desired RNA. Note that this gel uses staining for detection, so the DNA template and nontemplate strands are also present, as marked.

FIG. 8 illustrates a crosslinked transcription complex. T7 RNA polymerase containing an N-terminal Strep-Tag®-II peptide and functional template DNA labeled with biotin at the 5′ end of the nontemplate strand are bound to (tetravalent) StrepTactin®XT magnetic beads.

FIG. 9 shows tethered, high salt transcription dramatically reduces primer extension. FIG. 9A shows salt dependence of transcription profiles for tethered and untethered complexes analyzed by 20%, 7M urea denaturing gel electrophoresis, labeled via incorporation of [α-³²P]ATP. The final concentrations NaCl added (to the standard reaction mixture) are shown. FIG. 9B shows quantification of individual gel lanes in 9A.

FIG. 10 shows tethered, high salt transcription does not impair transcription. FIG. 10A shows the salt dependence of transcription profiles for tethered and untethered complexes encoding RNA-24Alt, analyzed as in FIG. 2. FIG. 10B shows the quantification of individual lanes in FIG. 10A.

FIG. 11 shows Improvements are independent of RNA length. FIG. 11A shows the low and high salt transcription profiles for tethered and untethered complexes analyzed as in FIG. 9. FIG. 11B shows gel quantification of FIG. 11A.

FIG. 12 shows the system can be reused. FIG. 12A shows a summary of the repeated reaction cycle. FIG. 12B shows a low (0 M) and high (0.4 M) added NaCl transcription profiles for untethered and tethered transcription of RNA-24Alt analyzed as in FIG. 10. After “Use 1” the reaction solution was removed, beads washed, and a new substrate NTP solution was added to initiate “Use 2” (with the same cycle subsequently used to initiate “Use 3”). FIG. 12C shows gel quantification of the individual lanes in 10B.

FIG. 13 illustrates flow reactors for synthesis. FIG. 13A displays a diagram of a reactor in current development, shown in FIG. 13D. The precise nature of the coupling shown in FIG. 13B is likely not important. The inset FIG. 13C shows actual beads (not yet carrying RNA polymerase) being held in place by flow against a designed, physical barrier. The design in FIG. 13E is a simple extension of FIG. 13A, including a chamber with beads containing 3′-capture DNA (or with an affinity tag to bind 3′ capture DNA flowing continuously).

FIG. 14 shows an initial prototype flow chamber (left) alongside an example production device (right).

FIG. 15 shows the dramatic improvement in batch synthesis using enzyme and DNA tethered to a solid surface, modeling synthesis in a flow reactor.

FIG. 16 illustrates additional methods for coupling an RNA polymerase to a functional template DNA.

FIG. 17 illustrates alternate tethering schemes. FIG. 17A shows covalent attachment of the nontemplate DNA to a Halo-Tag® domain fused to the polymerase generates a “universal reagent.” Users provide template DNA encoding RNA of interest. FIG. 17B illustrates for long (e.g., mRNA) RNA, a Br-modified DNA duplex can be generated (via PCR), and then (covalently) bound to Halo-T7RP as above.

FIG. 18 shows that nicking the nontemplate DNA at position −4 yields a response representative of the expected stronger promoter binding: the salt resistance, relative to the native control, is increased and the reaction yields higher purity of the target RNA. Nontemplate to +2 (B) mimics full nontemplate (A) and shows significant salt sensitivity. Nontemplate to −5 (C) removes the barrier to melting, strengthening binding, which leads to salt resistance. Introduction of a nick at −4 by assembling three pieces of DNA (D) reduces the barrier to melting and confers salt resistance. A nick can also be introduced enzymatically (E) and confers the same resistance. The last example is important for applications using arbitrary length, PCR-generated DNA functional templates, as deoxyU can be introduced in the 5′ PCR primer.

FIG. 19 illustrates an affinity capture method for product RNAs. DNA with a component complementary to the 3′ end of the desired RNA selectively binds only RNAs with a free 3′ end, allowing for affinity chromatography or other affinity separation to purify only full length, single-stranded target RNA.

FIG. 20 shows rolling circle amplification of capture DNA. As shown in FIG. 20A, linear DNA containing the reverse complement (capture DNA) of the 3′product RNA sequence is ligated to form a circular DNA functional template. A primer is then extended using a strand displacing DNA polymerase to generate a long capture DNA containing repetitions of the capture sequence. As shown in FIG. 20B, the RCA construct can dramatically increase the capacity of any immobilization system. As shown in FIG. 20C, an RCA primer can attach to solid support for purification via a variety of means:

FIG. 21 illustrates an example of rolling circle amplification. FIG. 21A shows capture DNA circularized with ligase (either with or without a “splint” DNA to aid circularization). dT25 DNA is covalently attached to beads is then used to prime rolling circle amplification. The product is capture DNA covalently attached to magnetic beads that contains many target sites for purification. FIG. 21B shows the resulting bead-capture DNA conjugate is then used as in the method of FIG. 19 to purify only correct length RNA.

FIG. 22 shows transcription of site-specifically gapped mRNA encoding red fluorescence protein. FIG. 22A shows gel electrophoretic analysis. FIG. 22B shows an immunoblot analysis of the same samples using an antibody to dsRNA.

The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.

DETAILED DESCRIPTION

As illustrated in FIG. 1, under the current high yield synthesis conditions used in the production of mRNA, a side reaction, in which RNA polymerase binds and extends the accumulating product RNA, turns the correct RNA (or other RNAs) into double stranded RNA (dsRNA). Without being held to theory, it is believed that this dsRNA is the primary source of the immune responses that have been observed in RNA therapy. One of the primary barriers for mRNA therapeutics has been that the mRNA therapeutic has often triggered our “innate immune response.” This innate immune response has evolved as an early warning against viral infection, as many viruses that infect mammals have dsRNA genomes (or replication intermediates). Contaminating dsRNA in the therapeutic mRNA triggers this immune response, which manifests as inflammation. Using modified bases and extensive purification, companies have recently been able to lower the dsRNA levels enough for vaccines, but not yet enough for other mRNA therapeutics. The methods described herein provide an improved solution for the production of mRNA therapeutics, or RNAs of any length, with reduced immunogenicity and high purity.

As used herein, immunogenic dsRNA is RNA that is about 40 bases pairs in length. Complete base pairing is not required, allowing for mismatches and bulges. Immunogenic dsRNA is typically polydisperse, which can make analysis difficult. However, antibodies, such as monoclonal antibodies, which specifically bind dsRNA can be used to identify and quantify the dsRNA in an mRNA sample.

Based upon this understanding of how immunogenic dsRNA is made under high yield conditions, the inventors have developed multiple approaches to reducing dsRNA synthesis from the outset. The underlying principle is to prevent re-binding of the correct length RNA to the polymerase, preventing its extension to the double stranded form. All of the methods described herein are complementary to the use of modified nucleotides.

Some definitions are provided.

The term “polynucleotide” is interchangeable with nucleic acid and includes any compound and/or substance that comprise a polymer of nucleotides. RNA transcript products produced by the methods described herein and functional templates DNA used in the methods are polynucleotides. Exemplary polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof. Polynucleotides may include naturally occurring nucleotides as well as modified nucleotides such as pseudouridine 1-N-pseudouridine, dye-labeled nucleotides, biotin-labeled nucleotide, and others.

As used herein, an “RNA transcript” or “product RNA” refers to a ribonucleic acid produced by an in vitro transcription reaction using a DNA functional template and an RNA polymerase. The RNA transcript can include modifications, e.g., modified nucleotides. Product RNAs can have lengths of 10 to greater than 5,000 and include, for example, mRNAs.

A DNA “functional template” refers to a polynucleotide template for RNA polymerase. Typically, functional template DNA includes the sequence coding for a product RNA of interest operably linked to a fully or partially double stranded RNA polymerase promoter region. A preferred functional template DNA is double stranded, includes a template strand and a complementary non-template strand, and comprises a promoter region for a single-subunit, DNA-dependent RNA polymerase

“Operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like. For example, a coding sequence for a product RNA operably linked to an RNA polymerase promoter allows transcription of the product RNA.

Any number of RNA polymerases or variants may be used in the methods described herein. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, an SP6 RNA polymerase, a K11 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids, polymerases showing reduced abortive cycling, and polymerases with increased thermostability. As used herein, the terms a T7 RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, and an SP6 RNA polymerase include both wild type, mutant and truncated polymerases, so long as RNA polymerase activity is maintained.

RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence. As a non-limiting example, the RNA polymerase may be modified to exhibit an increased ability to incorporate a 2′-modified nucleotide triphosphate compared to an unmodified RNA polymerase (see International Publication WO2008078180 and U.S. Pat. No. 8,101,385; herein incorporated by reference in their entireties). Variants may be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and/or nucleic acid sequence and/or by using other methods known in the art.

“Conditions for product RNA synthesis” are in vitro transcription conditions. Such conditions comprise a transcription buffer, nucleotide triphosphates (NTPs), optionally an RNase inhibitor and an RNA polymerase if not explicitly defined. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.

“Conditions for DNA amplification” include a DNA polymerase such as a thermostable DNA polymerase, upstream and downstream primers if not explicitly defined, an amplification buffer, dNTPS and temperature/time conditions such as temperature cycling to produce an amplified DNA. When DNA is amplified, the upstream and downstream primers become incorporated into the amplified DNA.

An exemplary in vitro transcription reaction includes the following: an RNA polymerase, e.g., a T7 RNA polymerase at a final concentration of, e.g., 1000-12000 U/mL, e.g., 7000 U/mL; the DNA functional template at a final concentration of, e.g., 10-70 nM, e.g., 40 nM; nucleotides (NTPs) at a final concentration of e.g., 0.5-10 nM, e.g., 7.5 nM each; magnesium at a final concentration of, e.g., 12-60 mM, e.g., magnesium acetate at 40 mM; a buffer such as, e.g., HEPES or Tris at a pH of, e.g., 7-8.5, e.g. 40 mM Tris HCl, pH 8

In some embodiments, 5 mM dithiothreitol (DTT) and/or 1 mM spermidine is included in the transcription reaction. In some embodiments, an RNase inhibitor is included in the in vitro transcription reaction to ensure no RNase induced degradation during the transcription reaction. For example, murine RNase inhibitor can be utilized at a final concentration of 1000 U/mL. In some embodiments, a pyrophosphatase is included in the in vitro transcription reaction to cleave the inorganic pyrophosphate generated following each nucleotide incorporation into two units of inorganic phosphate. This ensures that magnesium, which is essential for transcription, remains in solution and does not precipitate as magnesium pyrophosphate. For example, an E. coli inorganic pyrophosphatase can be utilized at a final concentration of 1 U/mL.

The in vitro transcription reaction can be allowed to proceed, for example, under constant mixing at 37° C. for 1-12 hours. Typical yields can be, e.g., 5 mg of RNA per mL of transcription reaction.

In an aspect, a method of synthesizing a product RNA comprises

-   -   preparing a reaction mixture comprising a functional template         DNA for the product RNA, an RNA polymerase and a capture DNA         under conditions for product RNA synthesis,     -   synthesizing the product RNA in the reaction mixture,     -   optionally removing the capture DNA after product RNA synthesis,         and     -   optionally purifying the product RNA,     -   wherein the capture DNA is 3′ end protected, and is         complementary to 8 to 20 or more, preferably 12 to 20         nucleotides or more at the 3′ end of the product RNA, and     -   wherein the functional template DNA, the RNA polymerase, the         capture DNA or a combination thereof, is optionally covalently         or noncovalently attached to one or more solid supports.

The approach involves carrying out in vitro transcription in the presence of a 3′-end protected capture DNA oligonucleotide. The minimum length of the portion of the capture DNA that is complementary to the 3′ end of the product RNA is determined by stability. The use of modified nucleotides can increase stability at shorter lengths. In general, the overall length of the capture DNA is not critical so long as the complementary region has sufficient length to complex with the 3′ end of the product RNA. Thus, the capture DNA may be longer than the region that is complementary to the 3′ end of the product RNA.

This method dramatically favors correct, promoter-dependent transcription and prevents promiscuous promoter-independent activities. Using the capture DNA method, high yields of pure runoff RNA of various sizes can be produced. The method is tested to be superior to the traditional RNA synthesis methods (both synthetic and enzymatic) in terms of quality, scale, speed and price. The method has been compared with the commonly used HiScribe™ T7 High Yield RNA synthesis kit (New England Biolabs) and the capture DNA method was shown to be far superior in terms of quality and purity, as assayed by gel electrophoresis, while being comparable in scale, speed and price. The capture DNA method is complementary to the use of HiScribe™.

The functional template DNA, the RNA polymerase, the capture DNA or a combination thereof, is optionally covalently or noncovalently attached to one or more solid supports. In an aspect, the capture DNA and the functional template DNA and/or RNA polymerase are on separate supports, although it is possible for them to be linked to the same support.

Exemplary groups for 3′ end-protection of the capture DNA include 3′ amino, 3′-deoxy (H), 3′-phosphorylation, 3′-O-methyl, 3′-fluorophore, 3′-biotin, 3′-azide, 3′-fluoro, 3′-PEG, 3′-mismatched bases, and any modification that is inconsistent with the native phosphoryl transfer function of an RNA polymerase.

In an aspect, the capture DNA is removed by elevated temperature, DNase, or toehold-mediated strand displacement, for example.

In an aspect, the RNA is purified using commercially available kits such as the Ambion/Applied Biosystems (Austin, Tex.) MEGAClear RNA™.

In an aspect, the RNA polymerase is covalently or noncovalently linked to the functional template DNA, wherein the noncovalent linking allows for synthesis of the product RNA.

In a specific aspect, the RNA polymerase is covalently or noncovalently linked to a nontemplate DNA that is 10 to 10,000 nucleotides or longer in length, and wherein the nontemplate DNA is complementary to a minimum of 10, 14, preferably 17 nucleotides of the template DNA upstream of the transcription start site, or wherein the nontemplate DNA is complementary to all or part of the entire template strand DNA in the coding region. The nontemplate DNA noncovalently links the RNA polymerase to the template DNA by essentially acting as an intermediary between the RNA polymerase and the template DNA.

In another aspect, a method of synthesizing a product RNA comprises

-   -   preparing a reaction mixture comprising a functional template         DNA for the product RNA, and an RNA polymerase under conditions         for RNA synthesis, wherein the RNA polymerase is covalently or         noncovalently linked to the functional template DNA, wherein the         linking allows for synthesis of the product RNA,     -   synthesizing the product RNA in the reaction mixture, and     -   optionally purifying the product RNA,     -   wherein the functional template DNA, the RNA polymerase, or         both, is optionally covalently or noncovalently attached to a         solid support.

In an aspect, the template DNA is also covalently or noncovalently linked to the RNA polymerase by a means additional to the nontemplate DNA.

The template DNA can be linked to the polymerase by using 3′ modifications that parallel the 5′ modifications described herein. FIGS. 16 and 17 illustrate additional options for linking RNA polymerase to template and/or nontemplate DNA such as using a T7 RNA polymerase variant containing an N-terminal Strep-Tag® II peptide and a biotin labeled DNA template; using an aldehyde labeled T7 RNA polymerase and a biotin labeled DNA functional template; or using a halo labeled T7 RNA polymerase and a biotin labeled DNA functional template.

In another aspect a method of synthesizing a product RNA comprises

-   -   preparing a reaction mixture comprising a functional template         DNA for the product RNA, and an RNA polymerase under conditions         for RNA synthesis, wherein the RNA polymerase is covalently or         noncovalently linked to a nontemplate DNA that is 10 to 10,000         nucleotides or longer in length, and wherein the nontemplate DNA         is complementary to a minimum of 10 nucleotides of the template         DNA upstream of the transcription start site, or wherein the         nontemplate DNA is complementary to all or part of the entire         template strand DNA in the coding region,     -   synthesizing the product RNA, and     -   optionally purifying the product RNA,     -   wherein the template DNA, the nontemplate, the RNA polymerase,         or a combination thereof, is optionally covalently or         noncovalently attached to a solid support.

In this aspect, the nontemplate DNA noncovalently links the RNA polymerase to the template DNA by essentially acting as an intermediary between the RNA polymerase and the template for the RNA.

The methods include, for example, tethering template or nontemplate DNA and RNA polymerase to a solid support, or alternatively, simply to each other, and then optionally carrying out transcription at elevated salt concentrations. Elevated salt weakens the product re-binding that leads to longer, double stranded products. Tethering RNA polymerase and template DNA together, either directly or through a nontemplate DNA, allows the polymerase to function at these high salt concentrations. This method dramatically favors correct, promoter-dependent transcription and prevents promiscuous promoter-independent activities. The method can be used to synthesize high yields of pure runoff RNA of various sizes. The method has been compared side by side with the commonly used HiScribe™ T7 High Yield RNA synthesis kit (New England Biolabs) and was shown to be far superior in terms of quality and purity, as assayed by gel electrophoresis, while being comparable in scale, speed and price.

The template or nontemplate DNA and the RNA polymerase can be linked using a variety of bioconjugation chemistries such as those using amine, acid, hydrazine, aldehyde/ketone, hydroxylamine, maleimide/alkylhalide and sulfhydryl functional groups.

In an aspect, the RNA polymerase comprises a sequence for a formyl glycine generating enzyme, and the template or nontemplate DNA comprises a 5′ hydrazine which is covalently coupled to an aldehyde in the RNA polymerase.

In another aspect, the RNA polymerase comprises an avidin-binding peptide, the template or nontemplate DNA comprises a 5′biotin label, and the RNA polymerase is noncovalently linked to the template or nontemplate DNA via beads that bind both the avidin-binding peptide and the biotin.

An advantage of linking the RNA polymerase and the template DNA, either directly or through for example a nontemplate DNA, is that high salt conditions may be used for RNA production as well as low salt conditions. As used herein, high salt conditions are 50 to 1000 mM, preferably 100 to 500 mM. When a nontemplate DNA is employed, the nontemplate DNA acts as an agent that brings together the RNA polymerase and the template DNA. The nontemplate DNA also allows formation of the template-nontemplate duplex promoter sequence that is critical for binding in an initiation competent state.

In an aspect, the nontemplate DNA may include a tag, such as an affinity purification tag, such as biotin which binds to a streptavidin column.

In an aspect, the wherein contacting can further includes a capture DNA as described above in the first aspect.

In the foregoing aspects, the template DNA, the nontemplate DNA or the RNA polymerase, or all three, can be covalently or noncovalently attached to a solid support such as a bead via a linker or chemical moiety such as a polyethylene glycol linker. Exemplary solid supports include beads such as magnetic beads.

In another aspect, a method of synthesizing a product RNA comprises

-   -   (i) preparing a reaction mixture comprising a functional         template DNA for the product RNA, and an RNA polymerase under         conditions for RNA synthesis, wherein the functional template         DNA and the RNA polymerase are immobilized, e.g., tethered to a         solid support, and wherein the contacting is done in a flow         chamber,     -   synthesizing the product RNA and continuously removing the         product RNA from the flow chamber while flowing fresh RNA         synthesis reagents into the flow chamber, and     -   optionally purifying the product RNA,     -   or     -   (ii) preparing a reaction mixture comprising a functional         template DNA for the product RNA, and an RNA polymerase under         conditions for RNA synthesis, wherein the RNA polymerase is         covalently or noncovalently linked to a nontemplate DNA that is         10 to 10,000 nucleotides or longer in length, and wherein the         nontemplate DNA is complementary to a minimum of 10, 14,         preferably 17 nucleotides of the template DNA upstream of the         transcription start site, or wherein the nontemplate DNA is         complementary to all or part of the entire template strand DNA         in the coding region, wherein the nontemplate DNA and the RNA         polymerase are immobilized to a solid support, and wherein the         contacting is done in a flow chamber,     -   synthesizing the product RNA and continuously removing the         product RNA from the flow chamber while flowing fresh RNA         synthesis reagents into the flow chamber, and     -   optionally purifying the product RNA.

The approach involves tethering functional template DNA and RNA polymerase to a solid support such as a bead, and then carrying out transcription in a fluidics (flow) reactor, where product is continuously removed from the reactor, while fresh RNA synthesis reagents are flowed in. This process can be done under normal or elevated salt concentrations. This method should dramatically favor correct, promoter-dependent transcription and prevent promiscuous promoter-independent activities. This method will allow for the production of high yields of pure runoff RNA of various sizes.

Exemplary solid supports include microparticles and beads, for example. Enzyme and DNA can be attached to the beads via a variety of protocols (see above). In production, a complete system would comprise a multiple use device to deliver fluidics, mating with disposable “chips” on which the reaction is carried out. The device contains pumps, valves and automation to control flow rates and [optionally] delivered reagent concentrations. The disposable chip contains fluidics paths that encompass the reaction chamber, with or without pre-loaded bead-enzyme constructs onto which the desired functional template would attach (under setup flow). The chip mates appropriately with the device for flow. FIG. 11 shows the initial prototype, alongside an example production device. Variants can drive multiple chips in parallel, for higher throughput. Device/chip systems can be produced at different scales, to accommodate desired RNA synthesis yields.

In an aspect, the flow chamber is in operable communication with a downstream chamber comprising an immobilized reagent that specifically binds full-length product RNA, wherein the immobilized reagent does not bind less than full-length RNA and double-stranded RNA impurities.

In an aspect, the RNA polymerase is covalently or noncovalently linked to template DNA and/or a nontemplate DNA as described above. In this aspect, the RNA polymerase can comprise an avidin-binding peptide, the nontemplate DNA comprises a 5′biotin label, and the RNA polymerase is noncovalently linked to the nontemplate DNA via a bead support that bind both the avidin-binding peptide and the biotin.

The method can be conducted under low salt conditions of 0 to 50 mM, or high salt conditions of 50 to 1000 mM.

In another aspect, contacting further includes a capture DNA as described above.

In another aspect, the inventors have recognized that promoter binding that leads to synthesis of the desired RNA competes with the product rebinding that generates longer impurities. It has been previously shown that some of the energy of promoter binding is used to drive coupled melting of the DNA near the start site. Targeted “nicking” and/or “gapping” of the DNA in the nontemplate strand can increase promoter binding by the polymerase (for example, by reducing the barrier to DNA melting). A “nick” is a break in the phosphodiester backbone, wherein all of the sugars and nucleobases are present. An “abasic” site is a site in which a nucleobase is excised. A “gap” is a site in which the backbone is broken and one sugar and one nucleobase are missing. Removing the barrier to melting near the start site will increase promoter binding by the polymerase. Thus, a method of preparing an amplified functional template DNA is described herein.

A method of providing a modified, amplified functional template DNA comprises, providing a starting functional template DNA and an amplification primer, the amplification primer comprising one or more deoxyuridines on the nontemplate strand, under conditions for DNA amplification, amplifying the starting DNA functional template to provide an amplified DNA functional template, and excising the uracils from the amplification primer, providing the modified, amplified functional template DNA. Excising the uracils can be done using DNA glycosylase or mutants thereof. The modified, amplified functional template DNA has a weakened duplex in the melting region, which then strengthens promoter binding. The method optionally further comprises using exonuclease II to nick the modified, amplified functional template DNA. The resultant strengthening of binding will itself favor promoter binding over product re-binding, but then will also allow salt challenges. In direct analogy to the results of FIG. 5, the results presented in FIG. 12 demonstrate the predicted behavior: nicking the nontemplate DNA at position −4 significantly increases salt resistance, relative to the native control, and yields higher purity of the target RNA.

In an aspect, a method of synthesizing RNA comprises providing a starting functional template DNA for a single-subunit, DNA-dependent RNA polymerase, and first and second amplification primers for the starting functional template DNA, wherein the starting functional template DNA comprises a promoter region for the single-subunit, DNA-dependent RNA polymerase, wherein the first amplification primer is complementary to a template strand of the template DNA and comprises one or more deoxyuridine residues, wherein at least one of the one or more deoxyuridine residues is at position −1, −2, −3, −4 or −5 of a promoter region for the single-subunit, DNA-dependent RNA polymerase, and wherein the second amplification primer is complementary to a nontemplate strand of the template DNA; amplifying the starting functional template DNA in the presence of the first and second amplification primers and under conditions for DNA amplification to provide an amplified functional template DNA, wherein the amplified functional template DNA comprises the one or more deoxyuridine residues at position −1, −2, −3, −4 or −5 of the promoter region for the single-subunit, DNA-dependent RNA polymerase; excising the uracil bases of the deoxyuridine residues from the promoter region of the amplified functional template DNA to provide a modified, amplified functional template DNA; carrying out a transcription reaction to synthesize RNA from the modified, amplified functional template DNA in the presence of the single-subunit, DNA-dependent RNA polymerase and under conditions for RNA synthesis; and isolating the synthesized RNA from the transcription reaction.

In another aspect, a method of synthesizing RNA comprises providing a starting functional template DNA for an RNA polymerase and an amplification primer that is complementary to a template strand of the template DNA and comprises one or more deoxyuridine residues, wherein the starting functional template DNA comprises a promoter region for the RNA polymerase, and wherein at least one of the one or more deoxyuridine residues is at position −4 or −5 of a promoter region for the RNA polymerase; amplifying the starting functional template DNA in the presence of the amplification primer under conditions for DNA amplification to provide an amplified functional template DNA, wherein the amplified functional template DNA comprises the one or more deoxyuridine residues at position −4 or −5 of the promoter region for the RNA polymerase; excising the uracil bases of the deoxyuridine residues from the promoter region of the amplified functional template DNA to provide a modified, amplified functional template DNA; carrying out a transcription reaction to synthesize RNA from the modified, amplified functional template DNA in the presence of the RNA polymerase and under conditions for RNA synthesis to provide a transcription reaction, wherein the polymerase is a T7 RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, or an SP6 RNA polymerase; and isolating the synthesized RNA from the transcription reaction.

Thus, in the methods, two amplification primers are used to amplify the starting functional template DNA for a single-subunit, DNA-dependent RNA polymerase, for example, using PCR techniques known in the art. The first or upstream amplification primer, which is complementary to a template strand of the template DNA and comprises one or more deoxyuridine residues in the promoter region for the single-subunit, DNA-dependent RNA polymerase, provides sites for nicking/gapping/excision in the amplified functional template DNA. Excising the uracil bases to provide a modified, amplified functional template DNA increases promoter binding by the RNA polymerase and also reduces the amount of immunogenic dsRNA produced in the transcription reaction. The second or downstream amplification primer is complementary to a nontemplate strand of the template DNA.

In a specific aspect, the first amplification primer comprises one deoxyuridine residue at position −1, −2, −3, −4 or −5 of the promoter region for the single-subunit, DNA-dependent RNA polymerase. In an aspect, least one of the one or more deoxyuridine residues is at position −4 or −5 of the promoter region.

While the RNA synthesis can be conducted under low or high salt conditions, high salt can reduce the amount of immunogenic dsRNA that is produced. In an aspect, the RNA synthesis is conducted under high salt conditions of 50 to 1000 mM. Increased salt has the advantage of decreasing primed extension production of dsRNA, but also can reduce overall transcription. The promoter modifications described above restore promoter binding, allowing the use of higher salt concentrations, for higher purity without decreasing yield.

In a further aspect, excising the uracil bases of the deoxyuridine residues comprises treating with DNA glycosylase, an exonuclease, or a combination thereof, to create a nick, break or gap in the nontemplate strand of the modified, amplified functional template DNA. For example, the USER® Enzyme is a combination of a uracil DNA glycosylase and a DNA glycosylase lyase endonuclease VIII. The uracil glycosylase catalyzes the excision of a uracil base, while the endonuclease cleaves the backbone 3′ and 5′ to the abasic site creating a gap.

Exemplary single-subunit, DNA-dependent RNA polymerases include a T7 RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, an SP6 RNA polymerase, or a Syn5 RNA polymerase. As used herein, the term single-subunit, DNA-dependent RNA polymerase includes functional mutants, fragments and derivatives of the polymerases.

The methods can be used to synthesize RNAs of any length including RNAs having a length of 10 to 10,000 bases, or 10 to 5,000 bases, 90-130 bases (CRISPR RNAs), 400-10,000 bases (mRNAs), 200-10,000 bases (lncRNAs), and any length in between such as 20 to 900, 30 to 800, 40 to 700 and the like. Exemplary long RNAs include mRNA, a CRISPR RNA, or a lncRNA.

Advantageously, wherein the isolated, synthesized RNA has at least a 2-fold, 5-fold or 10-fold reduction in binding to an immunogenic dsRNA-specific antibody compared to a control synthesized RNA synthesized from a control template DNA having a template strand identical to the template strand of the modified, amplified functional template DNA and a non-template strand that does not have a missing base, nick, break or gap in the promoter region. Exemplary immunogenic dsRNA-specific antibodies include Anti-dsRNA monoclonal antibody J2, Anti-dsRNA monoclonal antibody K1, and Anti-dsRNA monoclonal antibody K2 available from Jenna Bioscience.

The foregoing approaches are expected to reduce the synthesis of impurities that are primer-extended RNAs longer than encoded by the DNA. However, impurities that are shorter than those encoded by the DNA can arise from an expected decrease in stability of the elongation complex as it approaches the end of a DNA functional template or by “bumping” of a leading polymerase by a trailing polymerase. What is needed are approaches that can reduce both types of impurities. In an aspect, a short DNA oligonucleotide complementary to the 3′ end of the product RNA can be used to affinity purify product RNAs that are full length and not double-stranded at the end, thus reducing both contaminants.

In any of the methods disclosed herein, the functional template DNA can be a modified, amplified functional template DNA made by the method described above.

In an aspect, a method of purifying a product RNA, comprising

-   -   adding a capture DNA to a reaction mixture for producing the         product RNA, the reaction mixture comprising the product RNA, a         functional template DNA for the product RNA, and an RNA         polymerase under conditions for RNA synthesis; wherein the         capture DNA is 10 nucleotides or longer in length, and wherein         the capture DNA comprises a capture sequence complementary to a         minimum of 10 nucleotides of the 3′ terminus of the product RNA,         and wherein the capture DNA is immobilized to a solid support;     -   incubating the immobilized capture DNA and the reaction mixture         to provide binding of the capture DNA and the product RNA,     -   washing to remove unbound reaction components, and     -   eluting the product RNA from the immobilized capture DNA.

In an aspect, eluting the product RNA is done at an elevated temperature of 40 to 95° C., although other means of elution such as a chemical denaturant (e.g., urea) or toehold-mediated strand displacement are possible.

In an aspect, the capture DNA is immobilized to the solid support via a linkage such as a PEG linkage. Exemplary solid supports include beads such as magnetic beads, as well as chromatographic supports.

Since capture of a product RNA with a capture DNA is stoichiometric, the overall purification yield of the foregoing purification is limited by the number of available capture DNA binding sites. In an aspect, the capture DNA comprises a plurality of capture sequences. Moreover, a porous bead material may be counter-indicated for capturing mRNA length RNAs, as the size of the pores may not accommodate the large polymeric RNA. A rolling circle amplification strategy to prepare a capture DNA comprising a plurality of capture sequences may be used to overcome these issues.

Rolling circle amplification (RCA), shown in FIG. 20A, is used to generate capture DNA with repeating sequence elements. Thus, a single stranded capture DNA attached to a solid support might now harbor 10-100 binding elements to capture product RNA 3′ ends, as illustrated in FIG. 20B. As shown in FIG. 20A, linear DNA containing the reverse complement (capture DNA) of the 3′product RNA sequence is ligated to form a circular DNA functional template. A primer is then extended using a strand displacing DNA polymerase to generate a long capture DNA containing repetitions of the capture sequence. As shown in FIG. 20B, the RCA construct can dramatically increase the capacity of any immobilization system. As shown in FIG. 20C, an RCA primer can attach to solid support for purification via a variety of means: 1) beads with covalently attached dT25 are commercially available and can anneal to a dA25 sequence engineered into the RCA template; 2) using the same beads, the primer could contain free dA25 to allow binding to the beads, and 3) an arbitrary priming sequence could be attached to an affinity tag such as biotin, for capture on complementary solid support.

There are a variety of approaches to preparing an RCA primer that is attached, or can be attached, to solid supports. Three are shown in FIG. 2C. In (1) dT25 covalently attached to non-porous magnetic beads is commercially available, and all or part of the dT25 element could anneal to dA25 engineered within the RCA template to prime extension. In (2) a dA25 element in the RCA primer, but external to the cyclic template would provide for noncovalent attachment to the same beads with covalently attached dT25. In (3) the RCA primer could contain a more traditional affinity tag, such as biotin for capture by streptavidin-coated beads. These are but three of a variety of immobilization approaches that would be viable in such a system.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1: RNA 3′ End Sequestration Materials and Methods

Reagents. DNA oligonucleotides used as transcription functional templates and as capture DNAs, and synthetic RNA for self-primed extension reactions, were purchased from Integrated DNA Technologies (IDT). All ‘high yield’ transcription reactions were conducted using HiScribe™ T7 High Yield RNA Synthesis Kit (New England BioLabs). For self-primed extension reactions, T7 RNA polymerase was prepared and purified in house.

RNA self-primed extension reactions. Reactions with synthetic RNA, in the absence of promoter DNA, were conducted with 25 μM synthetic RNA in the presence of 0.5 μM T7 RNA polymerase and 0.4 mM each of guanosine triphosphate (GTP), cytidine triphosphate (CTP), adenosine triphosphate (ATP), and uridine triphosphate (UTP). Reactions were carried out at 37° C. for both 5 min and 4 h in a transcription buffer containing 15 mM magnesium acetate, 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 25 mM potassium glutamate, 0.25 mM ethylenediaminetetraacetic acid and 0.05% Tween-20. For self-primed extension reactions in the presence of capture DNA, DNA oligonucleotides, complementary to the 3′ end of RNA and containing 3′ amino modification were added to reaction mixtures to a final concentration of 25 μM.

Transcription reactions. All reactions were performed using partially single-stranded DNA constructs, in which the nontemplate DNA oligonucleotide extends downstream only to position +2 (1, 41). All ‘high yield’ transcription reactions were carried out in the presence of 2 μM each of nontemplate and template DNA oligonucleotides, 7.5 mM of each NTP, and 1.5 μL T7 RNA polymerase Mix™ (New England BioLabs) in an overall 20 μL reaction volume at 37° C. for 4 h (unless noted otherwise). High yield transcription reactions in the presence of capture DNA additionally contained 400 μM (unless noted otherwise) capture DNA. RNase Inhibitor Murine (New England BioLabs) was added to a final concentration of 1 U/μL in all reaction mixtures. Both self-primed extension and transcription reactions were heat inactivated at 70° C. for 5 min.

Gel electrophoretic analyses. Reaction products were analyzed with 20% polyacrylamide, denaturing (7 M urea) gel electrophoresis. For self-primed extension reactions, the 5′ end of the synthetic RNA was labeled by incubating 50 μM synthetic RNA with [γ-³²P] ATP (PerkinElmer) and 1 μL (10 Units) of T4 polynucleotide kinase (New England BioLabs) in a 10 μL reaction volume at 37° C. for 30 min. Labeling was inactivated by heating at 65° C. for 20 min. Transcribed RNAs were labeled by including [α-³²P] ATP (PerkinElmer) in the reaction mixture (without reducing the concentration of ATP). All gel experiments were repeated at least twice and showed no significant differences. Quantifications of gel data, using ImageJ v1.52a on unprocessed and uncompressed TIF output.

Results

The determination that 3′ end additions arise from (transient, active site bound) RNA structures in which the 3′ end of the nascent RNA folds back on itself to prime extension, points to a potential intervention to competitively inhibit this process. The addition to the reaction of a short capture DNA complementary to the 3′ end of the expected runoff RNA should drive formation of an RNA-DNA hybrid, as shown in FIG. 2, inhibiting self-primed extension. However, the possibility exists that RNA polymerase might in turn carry out primed synthesis from the 3′ end of the capture DNA, generating partially chimeric double stranded impurities. To prevent such 3′ extension of the DNA, for all capture DNAs used here we replaced the 3′ sugar hydroxyl of the 3′ terminal base with an amino group. While other 3′ sugar modifications should similarly prevent nucleotide addition (phosphoryl transfer), 3′ amino modification of DNA is commercially available during synthesis, at minimal added expense.

Self-primed extension of synthetic RNA is inhibited by 3′ complementary capture DNA: Our previous work demonstrated self-primed extension of a specific 24-base RNA, synthesized as runoff from a DNA functional template or synthesized chemically. In that earlier work, we demonstrated very efficient self-primed extension from synthetic RNA, in the absence of T7 promoter DNA. The (same) synthetic RNA construct shown in FIG. 2A (25 μM) was incubated with T7 RNA polymerase and 0.4 mM of each NTP for 0 min, 5 min and 4 h at 37° C. The results shown in FIG. 2B in the absence (−) of 25 μM capture DNA show that in a 5 min reaction the synthetic RNA readily extends to longer RNA products that are chased to still longer products over 4 h, as observed previously.

As predicted, the results in FIG. 2B show that in the presence (+) of 25 μM of the 17-base capture DNA (FIG. 2C), self-primed extension is dramatically inhibited. This demonstrates that the presence of 3′ complementary single stranded capture DNA is an effective way to competitively prevent 3′ end additions.

Effect of varying the length of capture DNA in self-primed extension: The above data confirm that 17 bases of complementarity provides sufficient target affinity for maximal competitive binding. How short can capture DNA be to still effectively compete with functional binding of the free RNA to RNA polymerase? To test the effect of the length of capture DNA, we conducted self-primed extension reactions with 25 μM synthetic RNA in the presence of 25 μM capture DNA oligonucleotides with lengths of 14, 11 and 9 bases and compared them with 17-base capture DNA. All capture DNAs were designed to hybridize to the RNA from its 3′ end, included a 3′ amino modification, and were added to the reaction in equal concentrations to the synthetic RNA.

As shown in FIG. 3, a 9-base capture DNA (Capture-9) is unable to compete well with self-primed extension; the product profiles at both 5 min and 4 h are similar to those in the reaction containing no capture DNA. In contrast, the 11-base capture DNA (Capture-11) limits self-primed extension in a 5 min reaction but shows significant self-primed extension in a 4 h reaction. To a first approximation, at 37° C., the 14-base capture DNA (Capture-14) functions about as well as the 17-base capture DNA (Capture-17) in preventing unwanted self-primed extension.

Capture DNA prevents self-primed extension during high yield transcription: We have previously shown that, as commonly carried out, when pushing transcription reactions to high yield conditions (for example, 7.5 mM each NTP, and 4 h incubation at 37° C.), the desired runoff RNA can be a minority of the product. The high concentration of runoff RNA drives its rebinding to the polymerase to effectively compete with de novo initiation, and through the mechanism described above, generates n+1, n+2 and substantially longer RNA products. Just as a 3′ complementary capture DNA can block rebinding; we expect that it should also inhibit rebinding from competing with initiation during transcription. To test this expectation, we added excess capture DNA to an in vitro transcription reaction with functional template DNA encoding the above 24-base RNA (with the same 3′ end as above, and with only a slight difference in the sequence from position +4 to +6).

The results shown in the lanes labeled “24” in FIGS. 4A and 4B demonstrate that, as expected, the presence (+) of 400 μM 3′ complementary capture DNA during a 4 h, high yield transcription reaction dramatically reduces formation of primer-extended products relative to the reaction in the absence of capture DNA (−). Encoded expected length RNA product increases substantially, while primer-extended products are reduced dramatically.

We previously observed that not all RNAs efficiently prime self-extension. To confirm that the presence of capture DNA does not interfere with synthesis of RNAs with low propensity to form primer extended products, we carried out identical transcription reactions on a functional template encoding RNA-24Alt that has been shown not to generate large amounts of longer RNA products. The results presented in FIG. 4 in the lanes labeled “24Alt” confirm similar transcription profiles in both the presence (+) and absence (−) of capture DNA complementary to 24Alt RNA, confirming that capture DNA has no negative effect on transcription efficiency.

Generalizability of the 3′ capture DNA—length and sequence of the RNA: The RNAs synthesized above are short, allowing high resolution in the electrophoretic analysis of primer extended RNA products. The capture DNA at 17 bases, however, is only a bit shorter than the 24-base RNA itself. In order to extend the observation to longer length RNA, we used the same 17-base 3′ complementary capture DNA to sequester a RNA with the same 3′ terminal sequence, but with a 10 base insertion in the upstream, uncaptured region of the RNA, yielding a 34-base RNA, labeled “34” in FIG. 3. The results shown in FIG. 4 are essentially identical to the results on the shorter 24-base RNA, indicating the expected inhibition of self-primed extension in the presence of the complementary capture DNA.

Moreover, to confirm that this approach can be used as a general method to prevent self-primed extension we tested the effect of capture DNA for transcription on a functional template encoding a 24-base RNA with a different sequence, labeled “24B” in data not shown. The results show that addition of capture DNA to the transcription mixture can be used as a general method to inhibit self-primed extension for functional templates encoding different RNA sequences.

Titration of capture DNA oligonucleotide: The above experiments were carried out with 400 μM capture DNA in solution on the assumption that the RNA would not accumulate to levels higher than 400 μM. To examine the effect of the concentration (total amount) of capture DNA, we conducted transcription reactions under high yield condition in the presence of concentrations of capture DNA ranging from 400 μM down to 100 μM. The results presented in FIG. 5A, show that while 400 μM capture DNA dramatically reduces RNA 3′ extension, reducing capture DNA concentration to 300 μM, 200 μM and 100 μM allows for increasing self-primed extension. These results are consistent with the expected stoichiometric titration of capture DNA by increasing product RNA. As product RNA concentrations exceed that of capture DNA, residual free RNA now rebinds to RNA polymerase and primes extension.

A more subtle interpretation might be that lower concentrations of capture DNA yield lower fractional formation of RNA-DNA capture complex, due to incomplete binding. To test this, we carried out transcription reactions in the presence of 200 μM capture DNA as a function of time. Consistent with the limiting stoichiometry model, at the lower reaction times of 5 and 20 min (and therefore lower RNA concentrations), this lower amount of capture DNA nevertheless effectively inhibits self-primed extension (FIG. 5B). The results confirm that the concentration of capture DNA (of tight binding length and sequence) needs only to exceed the final concentration of RNA synthesized in the reaction.

Discussion: Formation of longer RNA products during the synthesis of expected runoff RNA has been reported by various research groups over the years. The origin of these undesired longer RNA products synthesis has been attributed to different mechanisms, including turn-around synthesis, in which RNA polymerase switches to the nontemplate strand at the 5′ end of the DNA template, nontemplated addition, and primer extension. In recent work using RNA-Seq to characterize products of transcription, we have shown that cis self-primed extension is the main mechanism leading to synthesis of these unexpected RNA products. In cis self-primed extension, as the runoff RNA accumulates, it can rebind to the RNA polymerase in a mode wherein it folds back on itself, priming transcription from its own upstream sequence as the template. Self-primed extension of the runoff RNA leads to a reduction in the yield of the expected RNA product, but more importantly, yields partially double stranded RNA impurities that may interfere with its applications (e.g., triggering the innate immune response in therapeutic applications).

In order to eliminate self-primed extension during high yield synthesis, we have introduced a new approach involving the addition to the reaction of a short capture DNA that is complementary to the 3′ end of the expected runoff RNA. Hybrid duplex formation effectively sequesters the RNA 3′ end, preventing it from looping back on itself (or binding in trans to another RNA) to prime RNA-templated extension. Furthermore, in order to exclude the possibility that RNA polymerase uses this capture DNA as a primer, we introduced a minor and inexpensive modification to capture DNA: replacement of the 3′ hydroxyl group by an amino group in the current study, but other modifications should readily serve the same function.

Model studies with synthetic RNA. As reported previously, incubation of synthetic RNA, RNA polymerase, and substrate NTPs yields substantial self-primed extension. In contrast, the results presented in FIG. 2 show that the presence of a 1:1 ratio of 17-base capture DNA leads to a dramatic reduction in the formation of primer extended products.

Effective competition by 3′ capture DNA should depend on the affinity of capture DNA for the 3′ end of the RNA. Very approximate predictions suggest that 17-base capture DNA should bind to its target RNA with a (free in solution) T. of about 38° C. Shortening capture DNA to 14, 11, and 9 bases will weaken affinity, as reflected in the predicted melting temperatures. Not surprisingly, the results in FIG. 3 demonstrate that capture DNA complementary to only the 3′ terminal 9 bases does not inhibit self-primed extension significantly. A 3′ complementary 11-base capture DNA shows evidence of competition but is not effective at long reaction times. However, a 3′ complementary 14-base capture DNA does provide effective competition. While it may seem surprising that oligonucleotides with relatively weak predicted (in solution) affinities compete well, it must be remembered that competition is relative to hairpins with only 2-3 base pair (internal) stems. Without being held to theory, we have proposed that the latter functions in self-primed extension due to interactions within the enzyme binding cleft that stabilize otherwise weak solution structures. It is possible that capture DNA benefits from similar stabilization, but of course, does not extend because of its 3′ amino modification.

Application to transcription reactions. Extending the above to in vitro transcription reactions, we observed a similar inhibition of self-primed extension in the presence of capture DNA, without substantial inhibition of promoter-directed initiation (FIG. 4). While precise quantification of total RNA is complicated given the spread in product lengths, assays with a DNA template encoding RNA that does not undergo significant self-primed extension (24Alt in FIG. 4), does not show inhibition of promoter-directed synthesis by the appropriate 17-base capture DNA. This suggests that RNA-DNA hybrid binding to RNA polymerase is not overly strong, relative to promoter binding and de novo initiation.

To generalize these results to longer RNAs, we inserted 10 additional bases into the functional template DNA sequence encoding RNA-24, yielding a new construct encoding a 34-base RNA (RNA-34). The results shown in FIG. 4 yield identical behavior: in the absence of capture DNA, self-primed extension predominates, and in the presence, it is dramatically reduced. We fully expect that any length RNA will benefit from this approach.

As an aside, this experiment also provides preliminary information on the possible lengths of self-primed extension. In our original study, self-primed extension showed a range of extended lengths, but rarely continued to the maximum theoretical length, corresponding to the 5′ end of the RNA. It could be that self-primed extension is fundamentally limited to short lengths, or alternatively, the functional complex may weaken as it nears the 5′ end of the templating RNA. The results with promoter DNA encoding a 34-base transcript strongly suggest the latter. Noting the compression of longer length RNAs inherent in gel electrophoresis (see, for example, the DNA standards in FIG. 4), it appears that self-primed extension on 34-base RNA is extending to longer added lengths than on 24-base RNA. This predicts that still longer RNA-RNA duplex formation will occur on still longer RNAs, and indeed other studies have observed very long extensions.

To demonstrate the sequence generality of this approach, we tested the effect of capture DNA on functional templates encoding different RNA sequence with high abundance of self-primed extension products. Results not shown indicate inhibition of self-primed extension in the presence of capture DNA complementary to the 3′ end of RNA.

It is expected in this competitive inhibition that when runoff RNA concentrations exceed that of capture DNA, the free residual RNA will now begin to prime self-templated extension. The results presented in FIG. 5A are consistent with this prediction, as lower concentrations of capture DNA during transcription allow for some self-primed extension during preparative-scale synthesis. Even 200 μM capture DNA allows for essentially complete inhibition of self-primed extension at short times/low RNA levels, but as transcription proceeds and RNA concentrations increase, self-primed extension begins to increase, as capture DNA is consumed.

Finally, most researchers will want to remove the capture DNA from the reaction and variety of approaches are common, as reviewed recently. The preferred solution will likely depend on the length of the RNA. While denaturing gel purification will readily separate product from capture DNA (and from enzyme, promoter DNA, and abortive products), for RNAs significantly longer than the capture DNA, simpler commercial kits exist.

Example 2: High Salt Synthesis

All protein-nucleic acid associations are sensitive to salt. Hence, in an approach that is independent, complementary and orthogonal to Example 1, increasing salt in the reaction can reduce/eliminate product RNA re-binding and extension as shown in FIG. 6. However, initial promoter binding, required for correct synthesis, also decreases significantly with salt. To restore binding, the functional template DNA was linked to the enzyme to ensure promoter binding, even in the presence of high salt (and to favor promoter-initiated transcription over other reactions, at all salt concentrations).

To achieve salt-resistant promoter-directed transcription, RNA polymerase was engineered to contain a recognition sequence for the formyl glycine generating enzyme, introducing a unique aldehyde near the upstream end of the promoter DNA (guided by crystal structures). Synthesis of nontemplate DNA containing a 5′ hydrazine, allows covalent coupling of the hydrazine to the aldehyde. This generates a “universal reagent” containing the nontemplate DNA tethered to the protein. A user can then add to the reagent template DNA containing DNA of fixed sequence upstream (to the left) of the transcription start site (indicated by an arrow in FIG. 7), with the desired RNA sequence encoded immediately downstream.

Transcription with this promoter-coupled enzyme now shows resistance to salt, allowing the desired, salt-dependent reduction in off-pathway reactions, while retaining promoter-directed transcription. As shown at 200 mM NaCl in FIG. 7, the result is dramatically more pure RNA relative to the untethered control. The “not crosslinked” controls demonstrate that without crosslinking, transcription is inhibited at 200 mM NaCl (or other salt).

In the above reaction, 100% coupling of the aldehyde-containing nontemplate DNA to the aldehyde containing protein has not been achieved. Higher overall yields can be achieved as the percentage of coupling is improved.

Example 3: High Salt In Vitro Transcription Using Promoter DNA and T7 RNA Polymerase Co-Tethered to Beads

The goal of this study is to eliminate RNA product rebinding and extension, while retaining promoter-directed transcription. Both initial binding of T7 RNA polymerase to its promoter and rebinding of product RNA are stabilized by electrostatic interactions between the positively charged residues on the RNA polymerase surface and the negatively charged phosphate backbone of the DNA or RNA. As a result, increasing salt concentrations should destabilize both promoter binding and product rebinding. We have previously shown that covalently crosslinking an engineered cysteine (A94C) in the N-terminal domain of T7 RNA polymerase to a 3′ thiol-modified template DNA creates a locally high concentration of the promoter near its binding site, allowing promoter binding, even at high salt. Initiation proceeds well and at least some of these complexes transition to a stable elongation complex. Elongation complexes of T7 RNA polymerase are stabilized by a topological locking of the RNA around the template DNA in the enzyme active site and so are resistant to added salt concentrations up to at least 0.2 M NaCl.

In order to tether the polymerase to the promoter DNA and still allow functional initiation and substantial transition to elongation, we bound them each to Strep-Tactin®XT magnetic beads. The N-terminal domain of T7 RNA polymerase (together with a hairpin loop from the C-terminal domain) forms the promoter binding platform and many N-terminal fusions of T7 RNA polymerase function well in promoter-directed transcription. Thus, we fused the Strep-Tag® II peptide (WSHPQFEK; SEQ ID NO: 8) followed by a short and flexible peptide linker (GGS), to the N-terminus of recombinant T7 RNA polymerase. The Strep-Tag® II peptide has nanomolar binding affinity to the specifically engineered Strep-Tactin®XT magnetic beads. We also prepared 5′-biotinylated nontemplate DNA (extending upstream from position −25 to position +2 downstream). Biotin is reported to have picomolar binding affinity to Strep-Tactin®XT magnetic beads. SEQ ID NOs. 9-14 were used in the following experiments.

To confirm that the Strep-Tag®-II peptide addition at the N-terminus of T7 RNA polymerase does not affect transcription activity, we performed in vitro transcription reactions using Strep-tagged polymerase and promoter DNA encoding a 24mer RNA (RNA 24) under high yield solution conditions. A gel analysis (data not shown) demonstrates identical transcription profiles using T7 RNA polymerase and its Strep-tagged variant. This confirms that the addition of the Strep-Tag®-II peptide has no adverse effect on the activity of T7 RNA polymerase. Similarly, biotinylating the upstream end of the promoter has no effect on promoter function (data not shown). Finally, we tested these constructs for transcription activity at 0.4 M added NaCl, and as expected for the uncoupled species, observed complete inhibition of transcription in all constructs.

Tethered system favors promoter transcription at high salt: Having demonstrated that the DNA and protein modifications do not perturb promoter binding and transcription, we proceeded to test the tethered system for function. Given that at low salt the dissociation constant for duplex promoter binding by T7 RNA polymerase is ≈10 nM, we first pre-incubated the 5′-biotinylated nontemplate strand, template strand encoding a 24 base RNA and Strep-tagged T7 RNA polymerase at final equimolar concentrations of 0.8 μM. We then incubated the assembled promoter complex with beads coated with tetrameric Strep-Tactin® XT to form the tethered in vitro transcription system illustrated in FIG. 8.

We hypothesized that elevated salt concentrations weaken both on and off-pathway binding interactions, while tethering T7 RNA polymerase to the promoter should restore promoter binding by increasing the local concentration of promoter DNA compared to the free RNA in solution. To test the hypothesis with our tethered in vitro transcription system, we performed a comparative analysis of transcription between tethered and un-tethered systems as a function of increasing concentrations of NaCl. In order to see the direct effect on cis primed extension activity, we selected a functional template that encodes a 24mer RNA known to serve effectively in 3′ primer extension. The gel analysis presented in FIG. 9A shows that as added NaCl concentration is increased from 0 M to 0.4 M in 0.1 M intervals, all transcription activity decreases for the un-tethered system, and is negligible at 0.4 M NaCl. In the tethered system, promoter binding is resistant to increasing salt and transcription proceeds, while product RNA rebinding to polymerase is inhibited, reducing the formation of longer products. By 0.3-0.4 M NaCl, primer extended products are dramatically reduced. At 0.4 M NaCl, the overall yield starts decreasing but the purity of the encoded RNA is at its highest. Overall, high salt transcription in the tethered system shows much higher purity with increased yield of the correct length product

Synthesis of encoded RNAs is not impaired by tethering: Not all encoded RNAs participate in 3′ self-extension. To confirm that tethered transcription does not have an effect on the fundamental efficiency of promoter driven transcription, we repeated the above comparative analysis with a functional template encoding a 24mer RNA (RNA-24Alt) that is known not to participate substantially in 3′ primer extension reactions. The gel analysis presented in FIG. 10A shows the overall loss of yield in RNA transcription with increasing NaCl concentration, using un-tethered Strep-T7 RNA polymerase in solution. At 0.4 M NaCl (Lane 5), un-tethered transcription is mostly eliminated. FIG. 10A further shows that for the tethered system promoter driven on-pathway transcription is essentially unaffected by increasing salt concentrations up to about 300 mM added NaCl. The tethered enzyme is as efficient at producing encoded RNA-24Alt at 0.3M as it is at 0 M, as expected. In summary, the promoter driven transcription yields are not affected by high salt concentrations in the tethered in vitro transcription system. Users can define whether they would like to use 0.3 M or 0.4 M salt depending on their desire for yield versus purity.

Generality of the system: The results presented in FIGS. 9 and 10 use DNAs encoding 24 base RNAs. To test the generality of this system, we took the 24mer RNA introduced in FIG. 9 and inserted 10 bases at position +8 (data not shown). Paralleling the experiment of FIG. 9, we compare in FIG. 11 un-tethered and tethered transcription of RNA-34 at low (0 M) and high salt (0.4 M) concentrations. As predicted by the general model, the tethered in vitro transcription system produces only the encoded 34mer RNA at high salt. This result confirms that the system can be used for RNA of longer length to transcribe high yields of encoded RNA while preventing the formation of self-extended longer RNA impurities.

Biotinylated DNA and Strep-T7 RNA polymerase bind at adjacent sites on the tetrameric Strep-Tactin®magnetic beads: To encourage both enzyme and DNA to attach to the same tetramer in the above experiments, we preincubated (at low salt) tagged polymerase and labeled promoter DNA before adding the Strep-Tactin® XT magnetic beads. Following assembly, a high salt wash was used to remove components not strongly bound to the beads. As controls, we prepared tethered transcription complexes using DNA and T7 RNA polymerase with only one or neither of the two modifications. The resulting in vitro transcription reactions with DNA encoding RNA-24 under high yield conditions (data not shown) confirm that the absence of one or both of the modifications destroys RNA synthesis, both at low and high salt concentrations.

Despite the high affinity of T7 RNA polymerase for its promoter, we expected that some free enzyme or DNA would bind to the beads independently. Without a partner, these should be inactive in transcription. To test for enzyme immobilized without a promoter partner, we challenged an assembled and washed system encoding RNA-24Alt by introducing in solution unmodified promoter DNA encoding RNA-34Alt. At low salt, RNA polymerase without a partner will bind the free DNA encoding 34mer and transcribe it. Data not shown demonstrates this hypothesis to be correct. While at low salt, both 24Alt and 34Alt are transcribed at levels similar to that of untethered transcription, at high salt, only tethered RNA-24Alt is transcribed.

The system is stable and reusable: In this system, Strep-T7 RNA polymerase and functional template DNA are immobilized on Strep-Tactin® XT magnetic beads. This suggests that this bead-immobilized transcription complex could be re-utilized for multiple rounds of transcription. To test this, we carried out one round of transcription as above and stopped the reaction by the addition of EDTA to 50 mM. As illustrated in FIG. 12A, the beads containing immobilized transcription complex were then separated from the supernatant (containing RNAs, NTPs, and transcription byproducts) using a magnetic stand. After resuspension in [wash/transcription] buffer, they were stored overnight at 4° C. The following day, the beads were washed again in wash buffer and then resuspended in transcription buffer containing fresh NTPs to initiate a second round of transcription. This cycle was repeated a third time. The results from all three rounds of transcription are compared in FIGS. 12B and 12C. Although there is some loss in activity with each cycle, the system is reasonably robust.

The gel analysis presented in FIG. 12B compares the products at each reaction cycle, under both low (0 M) and high (0.4 M) added salt conditions. The results show that the system can be used at least three times, with only a small loss in yield, by simply washing the immobilized complex and introducing transcription buffer with fresh NTPs.

Discussion: Transcription in vitro by T7 RNA polymerase is widely used to synthesize RNA of diverse lengths and sequences, due to the promoter specificity and robust nature of this system. It is, however, known that under high yield synthesis conditions, this enzyme can generate extensive non-promoter specific activity that contaminates the product pool with other than encoded RNA products. The RNA field has long followed a two-step methodology in order achieve high yields of desired RNA: 1) high yield transcription using T7 RNA polymerase followed by 2) (sometimes extensive) purification of the encoded RNA using gel or chromatic purification methods. We have recently shown that the quest for high yield only exacerbates the production of undesired, longer products. As high concentrations of encoded RNA accumulate, T7 RNA polymerase rebinds the product RNA and extends it via primed extension, independent of the promoter. This leads to two problems: 1) heterogeneous longer than desired, (partially) double stranded RNA is produced and 2) since correct product RNA is extended to longer impurities, the net yield of the desired product decreases. In some cases, gel electrophoretic or chromatographic purification methods can address the first problem, but with further decreases in yield of the desired product. Purification approaches work best for relatively short RNAs, where the resolution of the purification is sufficient. Preparative separation of a target 300 base RNA from products extended by 20-30 bases, for example, is often beyond most approaches. With increased emphasis on mRNAs many kilobases in length, other solutions are required.

In an attempt to decrease non-promoter driven activities during transcription, we introduce a novel and exclusively promoter specific in vitro transcription method using bead tethered promoter DNA and T7 RNA polymerase under high salt conditions. The latter inhibits all polymerase-nucleic acid interactions. To restore only promoter specific transcription, we tether Strep-tagged T7 RNA polymerase and biotinylated promoter DNA to Strep-Tactin® XT beads, bringing the enzyme in close proximity to its promoter to drive promoter binding, even at high ionic strength.

While increasing the salt concentration leads to complete inhibition of all enzymatic activity in the untethered system, as shown in FIGS. 9 and 11, in the tethered system high salt inhibits the undesired cis-primed extension activity of T7 RNA polymerase, while allowing promoter-directed transcription. As expected, the production of primer extended products decreases, leaving higher amounts of the encoded length, unextended product. At sufficiently high concentrations of salt, even tethered transcription begins to decrease, again as expected. For these constructs, a practical optimum of about 0.3 M added NaCl provides a good trade off of purity vs yield. While these experiments have been carried out on relatively short RNAs, the almost identical behavior of 24 and 34 length encoded transcripts (FIGS. 9 and 11, respectively) argues that this result should be generalizable to any length RNA (e.g., mRNA and lncRNA), as the model would predict.

In an attempt to characterize the salt dependence of promoter-driven transcription with less dependence on primer extension, we used a sequence, 24Alt, previously observed to yield less of the resolvable primer extended products. The result in FIG. 10 that 0.1 M added NaCl leads to increased 24mer suggests that this construct (untethered) is, in fact, driving primer extension, but as observed in the gel, in a more dispersed heterogeneous way. Indeed, there is a detectable “smear” across lengths longer than 24 bases, which for the untethered system decreases concomitant with an increase in 24mer product at 0.1 M added NaCl. For the tethered system, this trend continues to at least 0.2 M added NaCl. Thus, primer extension, under these conditions, appears to be more sensitive to salt than promoter-directed transcription.

Examples 1 and 2 are orthogonal and so the approaches could be combined for added benefit. One could use tethered, high salt transcription in the presence of 3′ capture DNA, since a wealth of prior studies shows that binding of capture DNA to RNA should show very minor dependence on salt at these levels.

Local “tethering” of the promoter near its binding site, promoter binding (and transcription) persists, even at high salt. Thus, the re-binding that leads to dsRNA can be reduced, while maintaining correct transcription.

Crosslinking has been done by two completely different means, demonstrating that it is not the nature of the crosslinking, but the crosslinking itself that is achieving the outcome.

In one case, elevated salt was not needed. Without being held to theory it is believed that the crosslinking allows promoter binding to compete well against product rebinding, even at normal, low salt conditions. Fine tuning the linkage will enhance this effect.

Example 4: Flow Chamber

A flow chamber is being developed wherein polymerase and DNA are tethered to each other, but also to a solid support (beads), as in Example 3 and FIG. 13. In a flow system, product RNA is continuously removed from the reactor and so the product cannot be reused to form dsRNA. Without being held to theory it is believed that this approach will reduce unwanted side reactions. In addition, all by-products of the reaction, including pyrophosphate and abortive RNA transcripts are continuously removed from the reactor as new substrate NTPs are added. In conventional high yield transcription, both depletion of substrate and accumulation of byproducts such as pyrophosphate ultimately lead to cessation of transcription. A flow reactor could synthesize RNA, unattended, at a uniform rate for very long lengths of time.

This approach is orthogonal to Examples 1 and 2. Thus, if desired, a flow reactor could be run at elevated salt concentrations.

In a next generation device, shown in FIG. 13E, a downstream chamber containing immobilized beads could serve as an affinity capture mechanism to “fish” out only full length, single stranded RNA from the reactor effluent. RNA less than full length (lacking the 3′ target sequence) and RNA double stranded at its 3′ end will flow through. If 3′ capture DNA is included as input, it would contain an affinity tag orthogonal to tags used for tethering in the reactor beads.

The above reactors are “preparative” and could be scaled in size, as needed. High bead binding capacity argues that preparative reactions, which might normally be carried out in 0.5 mL reactions, could be carried out in 50 μL, or smaller, reactor chambers. Flow rates could be optimized to efficiently use substrate NTPs and, as noted above, the only limit to the run times would be capacity of the capture chamber (and the life of the enzyme; T7 RNA polymerase is very stable, generally). Such devices are very inexpensive to manufacture and could be readily mass produced (and parallel reactors would be simple to implement on a single chip). Pump systems designed to drive flow under computer control are readily available at relatively low cost. The flow chips may be disposable. As noted in Example 2, bead-coupled “universal reagents” could be produced (and stored) in bulk, and then partnered at run time with user-provided template strand DNA encoding the desired runoff RNA. Alternatively, double stranded RNA containing the appropriate 5′ modification could be prepared by PCR, using a universal modified primer.

FIG. 14 shows an initial prototype flow chamber alongside an example production device. FIG. 15 shows the dramatic improvement in batch synthesis using enzyme and DNA tethered to a solid surface, modeling synthesis in a flow reactor.

Example 5: Alternate Tethering Schemes

FIG. 16 illustrates alternate tethering schemes. FIG. 17 shows A) Covalent attachment of the nontemplate DNA to a Halo-Tag domain fused to the polymerase generates a “universal reagent.” Users provide template DNA encoding RNA of interest. B) For long (e.g., mRNA) RNA, a Br-modified DNA duplex can be generated (via PCR), and then (covalently) bound to Halo-T7RP as above.

Example 6: Strengthening Promoter Binding to Disfavor Off-Pathway Reactions

FIG. 18 shows that nicking the nontemplate DNA between positions −5 and −4 yields a response representative of the expected stronger promoter binding: the salt resistance, relative to the native control, is increased and the reaction yields higher purity of the target RNA. FIG. 18B shows that rather than assembling DNA from single stranded fragments, PCR generated, double-stranded DNA can be nicked between positions −5 and −4 after PCR. Incorporation of dU at equivalent position −4 in the PCR primer used to generate the double-stranded DNA provides a unique recognition signal for Thermolabile USER® (Uracil-Specific Excision Reagent) II Enzyme.

Example 7: Capture of Product RNA

An initial prototype for the capture of product RNA with a nontarget DNA, is shown in FIG. 19, where affinity purification targets (only) RNA containing the terminal, encoded sequence, in single stranded form. As shown in FIG. 19, nontarget DNA immobilized on a solid support contains a 5′ terminal sequences that is complementary to just enough of the 3′ end of the full-length product RNA to provide binding at low or room temperature. The support is then washed to remove truncated RNAs with a shortened capture sequence and ds RNAs, whose capture sequence is hidden within RNA structure. Finally, the full-length RNA can be released by elevated temperature.

In FIG. 19, the red region of the immobilized capture DNA is designed to match the 3′ terminal sequence of the RNA. The length of this capture sequence is designed to have a moderate melting temperature. Binding would occur below that temperature; release would occur above that temperature.

The region in blue could be an arbitrary linking sequence of DNA or might be a “tethering” linkage, such as polyethylene glycol. The intent is to provide some distance between the solid support and the capture sequence

Example 8: Rca Amplification of Capture Sequence

FIG. 21 illustrates an example of rolling circle amplification. FIG. 21A) capture DNA containing a sequence identical to the 3′ end of the desired RNA followed by dA25 is obtained commercially. The capture DNA is then circularized with ligase (either with or without a “splint” DNA to aid circularization). dT25 DNA is covalently attached to beads is then used to prime rolling circle amplification. The product is capture DNA covalently attached to magnetic beads that contains many target sites for purification. The above illustration shows 4 sites, but a much larger number is possible per DNA. In data not shown, lengths that are long enough to increase the viscosity of the solution noticeably have been achieved. For this example, a DNA 1,000 bases in length would contain about 24-25 binding sites for RNA. FIG. 21B) the resulting bead-capture DNA conjugate is then used as in the method of FIG. 19 to purify only correct length RNA.

While this example uses commercially available dT25 covalently attached to beads, but any sequence and any attachment approach should work equally well.

The design of the capture sequence can be such that RNAs truncated by as few as 1-3 nucleotides might be discriminated. Washes at increasing temperatures could allow for the discrimination of weakly from strongly bound RNAs.

An alternative purification approach, magnetic beads could be replaced with a conventional chromatographic support. The target sequence would then be designed to allow weaker (but still significant) binding to the target RNA. Purified RNA would then be separated via a more conventional approach, such as collecting fractions as they pass through the column. RNAs containing the target sequence for capture would move most slowly through the column and would elute last. Discrimination of fractions collected might allow distinguishing RNAs shortened by as few as 1 base (which might bind to the target DNA, but more weakly and so be retarded less in its migration). This latter approach might allow for even larger scale up, should that be desired.

Example 9: Synthesis of Long RNA and Verification of Reduced Levels of Immunogenic dsRNA

mRNA encoding the red fluorescent protein, using DNA synthesized by PCR amplification with an upstream PCR primer containing dU at position −4 of the T7 RNA polymerase promoter. The templated DNA was treated with the USER® II enzyme system (New England Biolabs) to generate a nick in the nontemplate strand at position −4 (removal of the entire base). In the control, the USER® II treatment was not used.

Both DNA samples were then transcribed at 37° C. for 1 hr, under “high yield” reaction conditions (30 mM Mg(II), 5 mM each of ATP, CTP, GTP and UTP, 0.4 μM T7 RNA polymerase, 0.4 μM DNA) at the concentrations of added NaCl indicated in FIG. 22 to prepare the approximately 800 nucleotide long RNA. Each sample was then cleaned with Monarch® RNA clean up kit (to remove DNA and nucleoside triphosphates) and quantified by absorbance at 260 nm. Samples were then analyzed by native agarose gel electrophoresis and using immunoblot analysis by spotting purified mRNA on a positively charged nylon membrane, blocking the membrane with TBS buffer containing non-fat skimmed milk and probing it with anti-dsRNA antibody, clone rJ2 (Millipore-Sigma) at 4° C. overnight. The blots were then conjugated with goat anti-mouse IgG2a cross-adsorbed secondary antibody, Alexa Fluor® 488 (ThermoFisher) for 1 hr and imaged with a FLA typhoon 9500 imager to determine double stranded RNA contamination. The amount of RNA analyzed in each blot/lane is indicated in FIG. 22.

The gel electrophoresis results in FIG. 22A clearly show the same salt resistance as observed for the synthesis of 34mer RNA oligonucleotides in FIG. 18, confirming that the enzyme-induced nick is strengthening promoter binding. Due to the length of the mRNA, it is not possible to resolve dsRNA, however, the immunoblot data shown in FIG. 22B reveal a significant reduction in dsRNA levels, as expected. As observed for shorter oligonucleotides, even at 0 M added NaCl, there is a reduction in dsRNA. As before, this is further reduced with increasing added NaCl. This approach should be generalizable to any length RNA/mRNA.

The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 

1. A method of synthesizing RNA, comprising, providing a starting functional template DNA for a single-subunit, DNA-dependent RNA polymerase, and first and second amplification primers for the starting functional template DNA, wherein the starting functional template DNA comprises a promoter region for the single-subunit, DNA-dependent RNA polymerase, wherein the first amplification primer is complementary to a template strand of the template DNA and comprises one or more deoxyuridine residues, wherein at least one of the one or more deoxyuridine residues is at position −1, −2, −3, −4 or −5 of the promoter region for the single-subunit, DNA-dependent RNA polymerase, and wherein the second amplification primer is complementary to a nontemplate strand of the template DNA; amplifying the starting functional template DNA in the presence of the first and second amplification primers and under conditions for DNA amplification to provide an amplified functional template DNA, wherein the amplified functional template DNA comprises the one or more deoxyuridine residues at position −1, −2, −3, −4 or −5 of the promoter region for the single-subunit, DNA-dependent RNA polymerase; excising the uracil bases of the deoxyuridine residues from the promoter region of the amplified functional template DNA to provide a modified, amplified functional template DNA; carrying out a transcription reaction to synthesize RNA from the modified, amplified functional template DNA in the presence of the single-subunit, DNA-dependent RNA polymerase and under conditions for RNA synthesis; and isolating the synthesized RNA from the transcription reaction.
 2. The method of claim 1, wherein the first amplification primer comprises one deoxyuridine residue at position −1, −2, −3, −4 or −5 of the promoter region for the single-subunit, DNA-dependent RNA polymerase.
 3. The method of claim 1, wherein at least one of the one or more deoxyuridine residues is at position −4 or −5 of the promoter region.
 4. The method of claim 1, wherein RNA synthesis is conducted under high salt conditions of 50 to 1000 mM.
 5. The method of claim 1, wherein excising the uracil bases of the deoxyuridine residues comprises treating with DNA glycosylase, an exonuclease, or a combination thereof, to create a nick, break or gap in the nontemplate strand of the modified, amplified functional template DNA.
 6. The method of claim 1, wherein the single-subunit, DNA-dependent RNA polymerase comprises a T7 RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, an SP6 RNA polymerase, or a Syn5 RNA polymerase.
 7. The method of claim 1, wherein the synthesized RNA has a length of 10 to 10,000 bases.
 8. The method of claim 1, wherein the synthesized RNA is an mRNA, a CRISPR RNA, or a lncRNA.
 9. The method of claim 1, wherein the isolated, synthesized RNA has at least a 5-fold reduction in binding to an immunogenic dsRNA-specific antibody compared to a control synthesized RNA synthesized from a control template DNA having a template strand identical to the template strand of the modified, amplified functional template DNA and a non-template strand that does not have a missing base, nick, break or gap in the promoter region.
 10. A method of synthesizing RNA, comprising providing a starting functional template DNA for an RNA polymerase and an amplification primer that is complementary to a template strand of the template DNA and comprises one or more deoxyuridine residues, wherein the starting functional template DNA comprises a promoter region for RNA polymerase, and wherein at least one of the one or more deoxyuridine residues is at position −4 or −5 of a promoter region for the RNA polymerase; amplifying the starting functional template DNA in the presence of the amplification primer under conditions for DNA amplification to provide an amplified functional template DNA, wherein the amplified functional template DNA comprises the one or more deoxyuridine residues at position −4 or −5 of the promoter region for the RNA polymerase; excising the uracil bases of the deoxyuridine residues from the promoter region of the amplified functional template DNA to provide a modified, amplified functional template DNA; carrying out a transcription reaction to synthesize RNA from the modified, amplified functional template DNA in the presence of the RNA polymerase and under conditions for RNA synthesis to provide a transcription reaction, wherein the polymerase is a T7 RNA polymerase, a T3 RNA polymerase, a K11 RNA polymerase, or an SP6 RNA polymerase; and isolating the synthesized RNA from the transcription reaction.
 11. The method of claim 10, wherein RNA synthesis is conducted under high salt conditions of 50 to 1000 mM.
 12. The method of claim 10, wherein excising the deoxyuridine residues comprises treating with DNA glycosylase or a mutant thereof.
 13. The method of claim 10, wherein excising comprises treating with exonuclease II to nick the modified, amplified functional template DNA.
 14. The method of claim 10, wherein the synthesized RNA has a length of 10 to 5,000 bases.
 15. The method of claim 10, wherein the RNA is an mRNA. 